Vanillin (4-hydroxy-3-methoxybenzaldehyde), the organoleptic compound of the vanilla flavor, is one of the quantitative most widely used flavoring agents worldwide. Its demand has long exceeded the supply by the botanical source Vanilla planifolia. At present, most of the vanillin is synthesized chemically from guiacol, which originates from fossile raw materials, and lignin, a component in waste material from the wood pulp industry (Ramachandra Rao and Ravishankar, 2000). However, the demand for this “nature-identical” vanillin, which is mostly used in the food and beverage industry, is shifted towards the “natural” vanillin due to a rising health- and nutrition-consciousness of the costumers. Thus, biotechnological production of “natural” vanillin becomes more and more important (reviewed by Krings and Berger, 1998; Priefert et al., 2001).
Efforts have been made to produce vanillin by in vitro cultured Vanilla planifolia cells (Davidonis and Knorr, 1991). A de novo synthesis was also implemented using genetically modified yeast strains (Hansen et al., 2009). The main focus, however, was put on the biotransformation using isolated enzymes or different prokaryotic microorganisms as whole cell biocatalysts (Havkin-Frenkel and Belanger, 2008; Berger, 2009).
Besides lignin and phenolic stilbenes, like eugenol, the biotransformation of ferulic acid to vanillin is the most intensively studied method to produce “natural” vanillin (reviewed by Rosazza et al., 1995; Priefert et al., 2001). The precursor ferulic acid (3-(4-hydroxy-3-methoxy-phenyl)prop-2-enoic acid), a hydroxycinnamic acid, is a highly abundant substance since it is a constituent of many plant cell walls (Ishikawa et al., 1963; Escott-Watson and Marais, 1992; Ishii, 1997; Oosterveld et al., 2000). Many different microorganisms have been evaluated for the production of vanillin from ferulic acid comprising recombinant strains of E. coli, Pseudomonas ssp., Rhodococcus ssp., Bacillus subtilis, Aspergillus niger, Pycnoporous cinnabarinus, Amycolatopsis ssp. and Streptomyces ssp. (Lesage-Meessen et al., 1996; Okeke and Venturi, 1999; Muheim and Lerch, 1999; Achterholt et al., 2000; Overhage et al., 2003; Peng et al., 2003; Plaggenborg et al., 2006; Yoon et al., 2007; Barghini et al., 2007; Hua et al., 2007; Di Gioia et al., 2010; Tilay et al., 2010; Fleige et al., 2013). However, in most cases vanillin yields were low and biotransformation reactions slow. The low yields can mostly be ascribed to the high toxicity of vanillin (Krings and Berger, 1998). Enhanced vanillin production with adsorbent resins improved the vanillin levels up to 19.2 gl−1, but the molar yield of about 43% was rather low (Hua et al., 2007). Other drawbacks were inefficient heterologous gene expression and plasmid instabilities. A focus was also set on prevention of further degradation of vanillin to vanillyl alcohol or vanillic acid (Stentelaire et al., 1997; Bonnin et al., 1999; Oddou et al., 1999; Civolani et al., 2000; Overhage et al., 2000).
Bacteria from the genus Pseudomonas show a broad metabolic versatility as they can use a wide range of aromatic molecules as sole carbon sources (Clarke, 1982). The ferulic acid catabolism in Pseudomonas sp. strain HR199, P. fluorescens BF13 and P. putida KT2440 occurs via a coenzyme A-dependent, non-R-oxidative pathway as depicted in FIG. 1 (Narbad and Gasson, 1998; Gasson et al., 1998; Overhage et al., 1999b; Plaggenborg et al., 2003; Calisti et al., 2008). First, ferulic acid becomes activated to feruloyl-CoA catalyzed by feruloyl-CoA synthetase (EC 6.2.1.34; encoded by fcs). Second, the CoA thioester is hydrated and cleaved to vanillin and acetyl-CoA catalyzed by enoyl-CoA hydratase/aldolase (EC 4.2.1.101; encoded by ech). The vanillin dehydrogenase (EC 1.2.1.67; encoded by vdh), oxidizes vanillin to vanillic acid which is further catabolized to protocatechuic acid by vanillate-O-demethylase (EC 1.14.13.82; encoded by vanAB). Overhage et al. (1999b) also proposed a second route over 4-hydroxy-3-methoxyphenyl-β-ketopropionyl-CoA and vanillyl-CoA catalyzed by enzymes encoded by PP_3355 (aat) and probably PP_3354.
A recent study has used a metabolic engineered strain of P. fluorescens for the production of vanillin from ferulic acid (Di Gioia et al., 2010). By deletion of the gene vdh for the vanillin dehydrogenase and by overexpression of the structural genes fcs and ech on a low-copy vector the authors were able to produce up to 8.41 mM vanillin from 10 mM ferulic acid which was the highest final titer of vanillin produced with a Pseudomonas strain so far.
The prior art approaches for the microbial production of vanillin still suffer from one or more of the following drawbacks: low conversion rate of ferulic acid, low molar yield of vanillin, significant by-product formation.
The problem underlying the present invention therefore was the provision of a method which avoids at least one of the above-mentioned drawbacks.